CN115087465A - Peptide-nanoparticle conjugates - Google Patents

Peptide-nanoparticle conjugates Download PDF

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CN115087465A
CN115087465A CN202080076664.XA CN202080076664A CN115087465A CN 115087465 A CN115087465 A CN 115087465A CN 202080076664 A CN202080076664 A CN 202080076664A CN 115087465 A CN115087465 A CN 115087465A
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dendrimer
peptide
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洪承杓
郑宇珍
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Wisconsin Alumni Research Foundation
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Abstract

Described herein is a nanoparticle system comprising a multivalent nanoparticle core comprising a plurality of β -hairpin peptides conjugated thereto. Pharmaceutical compositions and methods of making the nanoparticle systems are also included. Further included are methods of immunotherapy comprising administering the nanoparticle system to a subject in need thereof, such as a human cancer patient.

Description

Peptide-nanoparticle conjugates
Cross Reference to Related Applications
This application is a continuation of PCT/US2019/058463 filed on 29, 10, 2019 and claims priority to US provisional application 62/927,293 filed on 29, 10, 2019, which is incorporated herein by reference in its entirety.
Technical Field
The present disclosure relates to compositions and methods for delivering therapeutic beta-hairpin peptides.
Background
Due to their high affinity and selectivity for target materials, antibodies have been successfully used as binding ligands for many applications including immunotherapy. However, low thermodynamic stability and high manufacturing costs are inherent disadvantages of proteins and have been obstacles to their routine use. Furthermore, proteins containing many surface functional groups (e.g., amine, carboxyl, hydroxyl, and thiol groups) are often incompatible with site-specific chemical reactions, which limits their use in advanced nanotechnology applications. One potential approach to solving these problems is to achieve peptides with the useful properties of small molecules and proteins. Advances in peptide design strategies, such as library screening and structure-based molecular design, have facilitated the development of artificial peptides that are superior to proteins in terms of target binding. Furthermore, peptides can be synthesized by a cost-effective chemical method, i.e. solid phase peptide synthesis (one amino acid at a time) allowing easy and precise adjustment of the amino acid composition and macromolecular topology.
Although peptides are promising protein alternatives, short peptides generally have lower target binding affinity than proteins. Furthermore, peptides do not generally retain their native folded structure when separated from their proteinaceous environment, which can significantly affect their physicochemical properties.
There is a need for new compositions and methods for delivering beta-hairpin peptides.
Disclosure of Invention
In one aspect, the nanoparticle system includes a multivalent nanoparticle core comprising a plurality of β -hairpin peptides conjugated thereto.
In another aspect, a pharmaceutical composition comprises the nanoparticle system and a pharmaceutically acceptable excipient.
In another aspect, a method of making a nanoparticle system includes contacting a multivalent nanoparticle core comprising a plurality of reactive end groups with a composition comprising one or more β -hairpin peptides under conditions sufficient to conjugate the plurality of β -hairpin peptides to the multivalent nanoparticle core and provide the nanoparticle system.
In another aspect, the method of immunotherapy comprises administering the nanoparticle system to a subject in need thereof.
Drawings
FIG. 1 shows a schematic of the development process of a multivalent dendrimer-peptide conjugate as a PD-1/PD-L1 antagonist.
FIG. 2 shows the structure of PD-1, engineered PD-1, and different peptide structures targeting PD-L1. 1 is beta H 1 -wt sequence, SEQ ID NO 2 is β H 1 Mutant sequence, SEQ ID NO 3 is. beta.H 2 -wt sequence, and SEQ ID NO 4 is β H 2 -wt sequence. The binding surface is highlighted (banding).
FIG. 3 shows a size comparison between the β H2_ mt peptide, the G7 PAMAM dendrimer, and the PD-1/PD-L1 interface, indicating that the dendrimer surface contains multiple peptides separated by sufficient spatial distance to bind.
FIG. 4 shows SPR sensorgrams of the binding of G7- β H2_ mt (A), G7- β H2_ wt (B), G7- β H1_ mt (C), and fully acetylated dendrimer (D) to immobilized PD-L1 protein.
FIG. 5 shows SPR sensorgrams for binding of G7- β H2_ mt conjugate to PD-L1 using acetylated dendrimers of 90% (A), 80% (B) and 60% (C).
FIG. 6 shows the concentration-dependent binding kinetics of G7- β H2_ mt conjugate with PDL1, with quantitatively measured binding kinetics (k) a : an association rate constant; k is a radical of formula d : an off rate constant; k D : equilibrium dissociation constant). Curves A-D represent 45, 90, 180, 270nM, respectively.
FIG. 7 shows the concentration-dependent binding kinetics of aPD-L1 antibody to PDL1 with quantitatively measured binding kinetics (k) a : an association rate constant; k is a radical of d : an off rate constant; k D : equilibrium dissociation constant). Curves A-D represent 25, 50, 100, 200nM, respectively.
FIG. 8 shows concentration-dependent binding kinetics of free β H2_ mt peptide with PDL1, with quantitatively measured binding kinetics (k) a : an association rate constant; k is a radical of d : an off rate constant; k D : equilibrium dissociation constant). Curves A-D represent 17, 25, 33, 42. mu.M, respectively.
FIG. 9 shows the CD spectra of G7- β H2_ mt conjugate (A), β H2_ mt peptide (B), and fully acetylated dendrimer (C).
Figure 10 shows FTIR spectra of G7- β H2_ mt conjugate and its fourier self-deconvolution analysis, in which β -sheets and β -turns are labeled. Illustration is shown: FTIR spectra of β H2_ mt peptide (top) and fully acetylated dendrimer (bottom).
Figure 11 shows a schematic of the excluded volume effect that reduces the entropy cost of peptide folding.
Figure 12 shows the MD simulation results of the folding behavior of β H2_ mt when initially extended β H2_ mt was conjugated to G5 PAMAM dendrimer (β H2_ mt in bands, atoms in G5).
FIG. 13 shows the MD simulation results of the folding behavior of β H2_ mt when initially folded β H2_ mt was conjugated to G5 PAMAM dendrimer (β H2_ mt is in band, atom is in G5).
Fig. 14 shows the binding of f β H2_ mt to PD-L1.
FIG. 15 shows competition assays for f β H2_ mt/PD-L1 complex for G7- β H2_ mt (A), aPD-L1(B), β H2_ mt (C), and fully acetylated dendrimer (D).
Figure 16 is a graphical representation of G7- β H2_ mt conjugate binding to multiple PD-L1 proteins.
FIG. 17 shows fluorescence microscopy images of 786-O cells treated with G7- β H2_ mt for 1 hour (fluorescence from rhodamine, left; bright field image, right), scale bar: 50 μm.
FIG. 18 shows fluorescence microscopy images of MCF-7 cells treated with G7- β H2_ mt for 1 hour (fluorescence from rhodamine, left; bright field image, right), scale bar: 50 μm.
FIG. 19 shows a schematic of an immune checkpoint blockade resulting in an increase in interleukin 2(IL-2) secretion by Jurkat T cells.
FIG. 20 shows IL-2 secretion by Jurkat T cells co-cultured with 786-O and MCF-7 cells after treatment with different groups.
Figure 21 shows a schematic representation of immune checkpoint blockade leading to a decrease in chemotherapeutic resistance of oncogenic cells.
Figure 22 shows cancer cell viability following Doxorubicin (DOX) treatment demonstrating chemotherapy resistance of cancer cells when incubated with different groups.
FIG. 23 shows a β -sheet rich protein-protein interaction interface.
FIG. 24 shows the folding structure changes of peptides isolated from the protein environment.
FIG. 25 shows a schematic representation of the stabilization of the beta-hairpin by the Trpzip-DPC system.
FIG. 26 shows the peptide sequences and molecular structures of pL1 and pL1 TZ.
FIG. 27 shows the CD spectrum of pL 1.
FIG. 28 shows the CD spectrum of pL1 TZ.
FIG. 29 shows G4-pL1 TZ.
Figure 30 shows a schematic of the excluded volume effect.
FIG. 31 shows concentration-dependent SPR sensorgrams for PD-L1 binding to pL1 (left), pL1TZ (center) and G7-pL1TZ (right).
FIG. 32 shows fluorescence microscopy images of 786-O and MCF-7 cells treated with pL1, pL1TZ, and G7-pL1TZ for 1 hour (fluorescence from rhodamine, left; bright field image, right).
FIG. 33 shows the change in tumor volume over time in female Balb/c mice inoculated with the 4T1 cell line and treated with free IgG, free aPD-L1, G7-PMAM dendrimer-Cy-5, G7-PMAM dendrimer-pPD 1-peptide-Cy-5, or free pPD1 peptide.
FIG. 34 shows the change in body weight over time in female Balb/c mice inoculated with the 4T1 cell line and treated with free IgG, free aPD-L1, G7-PMAM dendrimer-Cy-5, G7-PMAM dendrimer-pPD 1-peptide-Cy-5, or free pPD1 peptide.
Figure 35 shows tumor bioluminescence of the mice of figures 33 and 34.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
Detailed Description
Described herein is a novel engineering method for beta-hairpin peptides (surface beta-hairpin peptides, S.beta.P) isolated from protein surfaces. Described herein are peptide-nanoparticle conjugates that conformationally stabilize S β ps by conjugating them to the nanoparticle backbone (scaffold), which additionally allows the peptides to exhibit multivalent binding effects. For example, dendrimer-peptide conjugates (DPC) allow stabilization of peptide structures with minimal modification of the peptide structures, particularly β -hairpin peptides. The β -hairpin peptide can be stabilized by covalent cross-linking. However, chemical modifications often complicate the peptide preparation process, often leading to a significant reduction in synthetic yield. The introduction of interchain non-covalent binding is another common strategy; however, it requires a large number of amino acid substitutions, which may affect the physicochemical properties of the peptide. The nanoparticle (e.g., DPC) platform described herein provides a novel approach to effectively antagonize and target β -sheet rich protein surfaces.
The use of peptide fragments on the surface of proteins is an effective method for exploiting protein functionality. In particular, β -hairpin peptides are promising because secondary structures are involved in myriad protein interactions. Based on conformational similarity, hairpin structures also have the potential to serve as antagonist platforms targeting β -sheet rich protein surfaces (e.g., PD-1/PD-L1 interface), where broad and flat geometries are generally not druggable for small molecule drugs (fig. 23). Because this surface is ubiquitous in protein-protein interactions (PPIs) and plays a key role in the progression of diseases associated with protein aggregation, modulation of PPIs mediated by β -sheet rich surfaces has been an important and challenging problem in drug research. However, peptide conformations were easily unstable when isolated from the protein environment (fig. 24), which greatly affected their target binding potency. The short circulation time of plasma and the susceptibility to digestion by proteases are other drawbacks of peptides, which limit their widespread use.
Advantages of the nanoparticle systems described herein include the use of nanoparticle carriers with high water solubility, biocompatibility, modifiable surface groups, and multivalency.
In embodiments, the nanoparticle system includes a multivalent nanoparticle core comprising a plurality of β -hairpin peptides conjugated thereto. The plurality of beta-hairpin peptides can include a plurality of identical beta-hairpin peptides or different beta-hairpin peptides conjugated to the same nanoparticle core. In specific embodiments, the multivalent nanoparticle core comprises a hyperbranched polymer, a dendrimer, a dendron, a mixed nanoparticle, or a micelle. For example, the multivalent nanoparticle core may be 3 to 150nm in diameter.
As used herein, hyperbranched polymers are multivalent particles that are polydisperse and irregular in branching and structure. In contrast, dendrimers have a very regular, radially symmetric generation structure. Dendrimers are monodisperse, spherical polymers that are generally prepared in a multistep synthesis compared to hyperbranched polymers. The dendrimer structure is characterized by a multifunctional core representing a center of symmetry, various well-defined radially symmetric layers of repeating units (generations) and terminal groups.
Hyperbranched polymers include polyesters, polyesteramides, polyethers, polyamides, polyethyleneimines, polyglycerols, polyglycolides, polylactides, polylactide-co-glycolides, polytartrate esters, and polysaccharides. Hyperbranched polyesters including Perstorp AB
Figure BDA0003624602240000061
The hyperbranched polyesteramide comprises DSM BV of the Netherlands
Figure BDA0003624602240000062
Polyglycerol produced by Hyperpolymers GmbH, and hyperbranched polyethyleneimineIncluding BASF AG
Figure BDA0003624602240000063
Hyperbranched polymers also include polycaprolactone and copolymers, such as poly (D, L-lactide-co-glycolide), and Degussa AG from Dynapo;
Figure BDA0003624602240000064
and
Figure BDA0003624602240000065
polyester compound produced by product series.
The preparation of hyperbranched polymers, such as hyperbranched polyglycerols, is well known in the art. For example, controlled anionic ring-opening multibranched polymerization of glycidol is carried out to form hyperbranched polyglycerol. The hyperbranched polyglycerol is then reacted with succinic anhydride in pyridine to provide carboxylic acid end groups through ester linkages. Once the functional group content on the hyperbranched polyglycerol was confirmed, the hydroxyl groups were further functionalized by the following scheme: hyperbranched polyglycerol-OH + N- (p-maleimidophenyl) isocyanate (PMPI, 10-fold molar excess) in DMSO or DMF at pH 8.5 to obtain hyperbranched polyglycerol-maleimide. Thus, hyperbranched polyglycerols possess both carboxyl and maleimide functional groups, which can be reacted with corresponding crosslinkers and chemical groups, or can be further derivatized to suit the particular functional groups available.
The amphiphilic hyperbranched polymer can form a micelle-like structure. The hyperbranched polymer may be an "imperfect" molecule in that it may include linear moieties and may be characterized by random or asymmetric branching. Hyperbranched polymers can be selectively modified to achieve a variety of functionalities on the surface, as well as being linked to functional components, such as linking carbon chains to add hydrophobicity, and primary amine groups to provide hydrophilicity and activation for subsequent modification.
Advantages of hyperbranched polymers include a small unit size (typically less than 60nm in diameter) and relatively simple synthetic procedures. Possible drawbacks include a broad size distribution, and possible difficulties in controlling surface modification of specific functionalities.
The term "dendrimer" as used herein includes, but is not limited to, molecular structures having an internal core, internal layers (or "generations") of repeating units regularly attached to and extending from the internal (initiator) core, and external surfaces having terminal groups attached to the outermost generations, where each layer has one or more branching points. Dendrimers have a regular tree-like or "starburst" molecular structure. For example, nanoparticle dendrimers are typically 3 to 10nm in diameter.
Each successive dendrimer generation may be covalently bound to the previous generation. The number of reactive groups of the core structure determines the n-directionality and defines the number of structures that can be attached to form the next generation.
The number of branches in the tree depends on the branching valency of the monomer building blocks (including the core). For example, if the core is a primary amine, the amine nitrogen will be divalent, giving 1-2 branching motifs.
Exemplary dendrimers are alkylated dendrimers, such as poly (amidoamine) (PAMAM), poly (ethylenimine) (PEI), polypropyleneimine (PPI), diaminobutylamine polypropyleneimine tetraamine (DAB-Am4), polypropylamine (POPAM), polylysine, polyester, pterene (iptycene), aliphatic poly (ether), aromatic polyether dendrimers, or a combination comprising one or more of the foregoing.
The dendrimer may have carboxyl, amine, and hydroxyl termini, and may be of any generation, including but not limited to generation 1 dendrimer (G1), generation 2 dendrimer (G2), generation 3 dendrimer (G3), generation 4 dendrimer (G4), generation 5 dendrimer (G5), generation 6 dendrimer (G6), generation 7 dendrimer (G7), generation 8 dendrimer (G8), generation 9 dendrimer (G9), or generation 10 dendrimer (G10).
The PAMAM dendrimers contain internal amide linkages which enhance their biodegradability, thereby increasing the tolerance with respect to therapeutic applications in humans. The surface includes polar, highly reactive primary amine groups. The surface of amino-functional PAMAM dendrimers is cationic and can be derivatized by ionic interaction with negatively charged molecules or by the use of many well-known primary amine covalent functionalizing agents.
When PAMAM dendrimers are used, generation 0 to 7 PAMAM dendrimers are typically used. For example, the hybrid nanoparticles may be formed from: generation 0 PAMAM dendrimer (G0); generation 1 PAMAM dendrimer (G1); generation 2 PAMAM dendrimer (G2); generation 3 PAMAM dendrimer (G3); generation 4 PAMAM dendrimer (G4); generation 5 PAMAM dendrimer (G5); generation 6 PAMAM dendrimers (G6) or generation 7 PAMAM dendrimers (G7). PAMAM is commercially available from a variety of sources, including Sigma-Aldrich (Cat. No. 597309).
Diaminobutaneamine polypropyleneimine tetramine (DAB Am4) is a polymer with a 1, 4-diaminobutane core (4-carbon core) and 4 surface primary amino groups. When forming hybrid nanoparticles from DAB-AM4 dendrimers, 0 to 7 generation DAB-AM4 dendrimers are typically used. For example, the hybrid nanoparticles may be formed from: generation 0 DAB-AM4 dendrimer (G0); generation 1 DAB-AM4 dendrimer (G1); generation 2 DAB-AM4 dendrimer (G2); a 3 generation DAB-AM4 dendrimer (G3); generation 4 DAB-AM4 dendrimer (G4); generation 5 DAB-AM4 dendrimer (G5); generation 6 DAB-AM4 dendrimer (G6) or generation 7 DAB-AM4 dendrimer (G7). DAB-AM4 is commercially available from a variety of sources, including Sigma-Aldrich (Cat. No. 460699).
The multivalent nanoparticles may be formed from one or more different dendrimers. Each dendrimer of the dendrimer complex may have similar or different chemical properties than other dendrimers (e.g., the first dendrimer may be a PAMAM dendrimer and the second dendrimer may be a POPAM dendrimer).
The dendron is a monodisperse wedge dendrimer moiety with multiple end groups and a single reactive function at the focus. For example, a dendron can be grafted to a surface, another dendron, or a macromolecule. Bis MPA (bis dimethylolpropionic acid) dendrons are available from Sigma-Aldrich.
As used herein, "micelle" refers to an aggregate of amphiphilic molecules in an aqueous medium, having an internal core and an external surface, wherein the amphiphilic molecules are predominantly oriented according to their hydrophobic portions forming the core and hydrophilic portions forming the external surface. A variety of monoclonal antibodies, peptides, proteins and small molecules can be covalently bound to the hydrophilic head group of the micelle and the nanoparticle covered with a plurality of conjugated ICI, resulting in stronger binding kinetics. Micelles are generally in dynamic equilibrium with the amphiphilic molecules or ions that form them, which are present in solution in a non-aggregated form. Many amphiphilic compounds and amphiphilic drug compounds are known to spontaneously form micelles in aqueous media above a certain concentration (referred to as the critical micelle concentration or CMC), including, inter alia, detergents, surfactants, amphiphilic polymers, lipopolymers (e.g., PEG-lipids), bile salts, single-chain phospholipids and other single-chain amphiphiles. The amphiphilic (e.g., lipid) component of the micelle does not form a bilayer phase, a non-bilayer mesophase, an isotropic liquid phase, or a solid amorphous or crystalline phase. The concept of micelles, and the methods and conditions for forming micelles, are well known to those skilled in the art. Micelles may coexist with lipid particles in solution.
Exemplary micelles include those described in U.S. patent No. 9,212,258, which is incorporated herein by reference for its disclosure of micelles comprising an amphiphilic dendron-coil. Each amphiphilic dendron-coil includes a non-peptidyl hydrophobic core-forming block, a polyester dendron, and a polyethylene glycol (PEG) moiety. Micelles comprising an amphiphilic dendron-coil are also known as "multivalent dendron conjugates" and "dendrimer-based nanomicelles (DNMs)".
The hydrophobic core-forming block of the micelle is non-peptidyl, i.e., the hydrophobic core-forming block is not a peptide. In some embodiments, the micelle comprises a single type of amphiphilic dendron-coil (i.e., the amphiphilic dendron-coils in the micelle all have the same three components). In some embodiments, the micelle comprises more than one type of amphiphilic dendron-coil (i.e., the three components of the amphiphilic dendron-coil in the micelle are different).
In some embodiments, the non-peptidyl hydrophobic core-forming block of the amphiphilic dendron-coil comprises Polycaprolactone (PCL), poly (lactic acid) (PLA), poly (glycolic acid) (PGA), or poly (lactic-co-glycolic acid) (PLGA). In some embodiments, the non-peptidyl hydrophobic core-forming block is PCL. In some embodiments, the PCL is poly (epsilon-caprolactone). In some embodiments, the non-peptidyl hydrophobic core-forming block is PLA. In some embodiments, the non-peptidyl hydrophobic core-forming block is PGA. In some embodiments, the non-peptidyl hydrophobic core-forming block is PLGA. The non-peptidyl hydrophobic core-forming block has a molecular weight including, but not limited to, a molecular weight of about 0.5kDa to about 20 kDa. In some embodiments, the non-peptidyl hydrophobic core-forming block is poly (. epsilon. -caprolactone) having a molecular weight of about 3.5 kDa. In some embodiments, the non-peptidyl hydrophobic core-forming block is a poly (. epsilon. -caprolactone) having a molecular weight of 14 kDa.
In some embodiments, the polyester dendrons of the amphiphilic dendron-coil include, but are not limited to, generation 3 to generation 5, i.e., generation 3 (G3), generation 4 (G4), or generation 5 (G5) polyester dendrons having an acetylene or carboxylate core. In some embodiments, the polyester dendron is a G3 dendron. In some embodiments, the polyester dendron is a G5 dendron. In some embodiments, the polyester dendron has an acetylene core. In some embodiments, the polyester dendron is a 3 generation polyester-8-hydroxy-1-acetylene bis MPA dendron. In some embodiments, the polyester dendron has a carboxylate core.
In some embodiments, the PEG moiety of the amphiphilic dendron-coil is a methoxy PEG (mPEG) moiety, an amino-terminated PEG (PEG-NH) 2 ) A moiety, an acetylated PEG (PEG-Ac) moiety, a carboxylated PEG (PEG-COOH) moiety, a thiol terminated PEG (PEG-SH) moietyMoiety, N-hydroxysuccinimide-PEG (PEG-NHS) moiety, NH 2 -PEG-NH 2 Moiety or NH 2 -a PEG-COOH moiety. In some embodiments, the PEG moiety has a molecular weight including, but not limited to, a molecular weight of about 0.2kDa to about 5 kDa. In some embodiments, the PEG moiety is an mPEG moiety. In some embodiments, the PEG moiety is an mPEG moiety having a molecular weight of about 2 kDa. In some embodiments, the PEG moiety is an mPEG moiety having a molecular weight of about 5 kDa.
In one embodiment, the polyester dendrons are covalently modified with linear hydrophobic polymers to help promote chain entanglement and intramolecular interactions, which aid in the self-assembly of the core-shell micelle and enable hydrophobic drug molecules to be loaded within the micelle. When micelles are administered in vivo, the PEG moiety forms a hydrophilic crown (corona) with non-fouling properties and provides an increased circulatory half-life.
Biologically important properties, such as biodegradability, circulatory half-life, targetability, pharmacokinetics and drug release, can be controlled by varying the three components (also referred to as the three polymer blocks) of the amphiphilic dendron-coil. Furthermore, the copolymer structure is flexible and can be easily manipulated by varying the molecular weight of each component to fine tune the Hydrophilic Lipophilic Balance (HLB). For example, various embodiments employ PCL, a polyester dendron, and PEG having molecular weights of 0.5-20kDa, G3-G5 (about 0.9-3.5kDa), and 0.2-5kDa, respectively. Thus, HLB (20M) H /(M H +M L ) Wherein M is H Is the mass of the hydrophilic block, and M L Is the mass of the lipophilic block) varies widely between 2.22 and 19.94.
When dendrons are copolymerized with hydrophobic linear polymers such as Polycaprolactone (PCL), poly (lactic acid) (PLA), poly (glycolic acid) (PGA) and poly (lactic-co-glycolic acid) (PLGA) in amphiphilic dendron-coil generations, tapered amphiphilic dendron-coils in turn have advantageous structural properties because they form self-assembled micelles, which are thermodynamically favorable, and have a highly packed PEG surface layer to increase blood circulation time. The thermodynamic stability of the formed micelle and the unique structure which is easy to adjust.
The nanocarrier system comprises a mixture of hyperbranched polymers and other biocompatible nanoparticles. For example, such mixed nanoparticles include dendrimer-liposome, dendrimer-PEG-PLA, dendrimer-exosome (exosome) mixtures, which combine the unique advantages of dendrimers (2-10 nm in diameter) and larger nanoparticles (50-200 nm).
Exemplary hybrid nanoparticles (also referred to as nanohybrids) include those described in U.S. patent No. 9,168,225, which is incorporated herein by reference for its disclosure of hybrid nanoparticles. In this embodiment, the hybrid nanoparticle is a particle in which the nanocore is surrounded or encapsulated in a matrix or shell. In other words, the smaller particles are among the larger particles. In certain embodiments, the mixed nanoparticle comprises a nanocore inside a liposome. In other embodiments, the nanocore is surrounded by a polymer matrix or shell (e.g., a polymer nanoparticle).
The maximum diameter of the nanocore is preferably 1nm to 50 nm. More preferably, the nanocore has a maximum diameter of 1 to 40nm, most preferably a maximum diameter of 3 to 20 nm. The nanocore can be analyzed by dynamic light scattering and/or scanning electron microscopy to determine the size of the particles. The nanocore may have any shape and morphology. Examples of nanocores include nanopowders, nanoclusters, nanocrystals, nanospheres, nanofibers, and nanotubes. The nanocore skeleton is easily expelled due to its nanoscale size. Thus, the nanocore scaffold employed need not be biodegradable, but in particular embodiments, the nanocore scaffold is biocompatible, i.e., non-toxic to cells. The scaffolds are "biocompatible" if their addition to cells in vitro results in less than or equal to 30%, 20%, 10%, 5%, or 1% cell death, and does not cause inflammation or other such unwanted adverse effects in vivo.
Exemplary polymer backbones include, but are not limited to, polyamides, polysaccharides, polyanhydrides, poly-L-lysine, polyacrylamides, polymethacrylates, polypeptides, polyethylene oxides, Polyethyleneimines (PEI), or dendrimers such as poly (amidoamine) (PAMAM) and PAMAM (ethylenediamine-EDA) dendrimers or modified forms thereof, such as hydroxylated, acetylated, or carboxylated forms of the polymer. Other exemplary polymer backbones are described, for example, in WO98/46270(PCT/US98/07171) or WO98/47002(PCT/US 98/06963). The multivalent polymer backbone molecule may have a configuration selected from linear, branched, forked, or star-shaped.
In some embodiments, at least a portion of the multivalent polymer backbone molecule may be hydrophobic. In some embodiments, at least a portion of the multivalent polymer backbone molecule may be hydrophilic. In another embodiment, a portion of the multivalent polymer backbone molecule may be hydrophobic, and a different portion of the molecule may be hydrophilic. In particular embodiments, the multivalent polymer backbone molecule is cationic. In other embodiments, the multivalent polymer backbone molecule is electrically neutral. In other embodiments, the multivalent polymer backbone molecule is anionic. One skilled in the art will recognize that a variety of starting materials may be selected to obtain a multivalent polymer backbone molecule that exhibits the desired properties.
In one embodiment, the shell is a liposome composed of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, soy phosphatidylcholine, lecithin, sphingomyelin, synthetic phosphatidylcholine, lysophosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine or phosphatidylserine, wherein the phospholipids may be modified with a long-circulating agent or cryoprotectant. In another embodiment, the shell is a nanoparticle of a polymer consisting of a polymer selected from the group consisting of poly (γ -L-glutamyl glutamine), poly (γ -L-aspartyl glutamine), poly-L-lactic acid, poly (lactic acid-co-glycolic acid), polyalkylcyanoacrylate, polyanhydrides, polyhydroxy acids, polypropyltcorydalis esters, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, [ N- (2-hydroxypropyl) methacrylamide ] copolymers, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polyureas, polyamines, polyepsilon caprolactone and copolymers thereof, wherein the polymer is modified or derivatized to enhance proteolysis resistance, improve circulation half-life, reduce blood glucose levels, and/or reduce blood glucose levels, Reduced antigenicity, reduced immunogenicity, reduced toxicity, improved solubility, or improved thermal or mechanical stability. In a particular embodiment, the shell is biodegradable. In certain embodiments, the multivalent polymer backbone is cationic and consists of polyamide, polysaccharide, polyanhydride, poly-L-lysine, polyacrylamide, polymethacrylate, polypeptide, polyethylene oxide, polyethyleneimine, poly (amidoamine) (PAMAM), or PAMAM (ethylenediamine-EDA).
Another hybrid nanoparticle is a dendrimer-exosome mixture as described in U.S. application serial No. 16/011,922. The dendrimer-exosome mixture is an exosome loaded with one or more nanoparticle dendrimers. As used herein, exosomes refer to vesicles having membrane structures secreted from a variety of cells. The exosomes have a diameter of about 25 to about 150 nm. The exosomes may express markers such as VLA-4, CD162, CXCR4, CD9, CD63, CD81, or a combination thereof. In one embodiment, the exosomes are derived from stem cells or tumor cells isolated from a subject (e.g., a human subject).
In one embodiment, the exosomes are derived from stem cells or tumor cells isolated from a subject (e.g., a human subject).
The stem cells include embryonic stem cells or adult stem cells, preferably adult stem cells. The adult stem cell may be, but is not limited to, a mesenchymal stem cell, a human tissue-derived mesenchymal stromal cell (mesenchymal stromal cell), a human tissue-derived mesenchymal stem cell, a pluripotent stem cell, or an amniotic epithelial cell, preferably a mesenchymal stem cell. The mesenchymal stem cells may be derived from, but not limited to, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, placenta, and the like.
In one embodiment, the stem cell is a mesenchymal stem cell. Mesenchymal Stem Cells (MSCs) can specifically target an inflammatory region common in cancerous regions, i.e. MSC tumor homing.
In another embodiment, the exosomes are isolated from tumor cells. Tumor cells actively produce, release and utilize exosomes to promote tumor growth.
Exosomes may be produced by isolating a tumor or stem cell from a subject, expanding the tumor or stem cell to provide an expanded cell population, culturing the expanded cell population, and isolating exosomes secreted from the expanded tumor or stem cell. The internal components can be removed from the isolated exosomes to provide so-called empty shell (gshost) exosomes, which are essentially empty containers for loading components such as nanoparticle dendrimers. In addition to the above features, patient-derived exosomes may also provide patients with non-immunogenic nanocarrier shells, providing an option for personalized medicine.
To allow conjugation of immune checkpoint inhibitors, in one aspect, multivalent nanoparticles are modified by reaction with alkyl epoxides, wherein the R group of the epoxide has 1 to 30 carbon atoms. In some embodiments, the alkyl epoxide reacts with an amino group present on the multivalent nanoparticle to form an alkylated multivalent nanoparticle.
The amine groups present on the multivalent nanoparticles provide reactive sites for a variety of amine-based conjugation reactions using coupling linkers, including but not limited to: dicyclohexylcarbodiimide, diisopropylcarbodiimide, N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide, 1' -carbonyldiimidazole, N-succinimidyl S-acetylthioacetate, N-succinimidyl-S-acetylthiopropionate, 2-mercaptoethylamine, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succinimidyl iodoacetate, succinimidyl 3- (2-pyridyldithio) propionate. In some embodiments, reactive esters are used to connect multivalent nanoparticles and other compounds through ester linkages. Examples of reactive esters include, but are not limited to, N-hydroxysuccinimide ester, N-hydroxysulfosuccinimide ester, N-gamma-maleimidobutyryl-oxysulfosuccinimide ester, nitrophenyl ester, tetrafluorophenyl ester, pentafluorophenyl ester, thiopyridyl ester, thionitrophenyl ester. Preferably, the reactive ester group is an N-hydroxysuccinimide ester.
The nanoparticles described herein comprise a β -hairpin peptide, e.g., a β -hairpin peptide having a high affinity for checkpoint inhibitor receptors.
Exemplary β -hairpin peptides include tumor targeting peptides, such as peptides that bind to cell surface receptors, peptides that target intracellular receptors, and peptides that interact with the extracellular matrix. For example, tumor targeting peptides that bind to cell surface receptors include peptides that bind to integrins (e.g., α v β 3 integrin with RGD binding motif and α v β 6 integrin expressed on the surface of colon, liver, ovarian, pancreatic and squamous cancer cells). Other targets for tumor targeting peptides include aminopeptidase N, peptide transporter 1, epidermal growth factor receptor, prostate specific membrane antigen, mucin 1, urokinase plasminogen activator receptor, gastric release peptide receptor, somatostatin receptor, cholecystokinin receptor, neurotensin receptor, transferrin receptor, vascular endothelial growth factor receptor, insulin, ephrin receptor, and the like. Tumor targeting peptides that bind to intracellular receptors include peptides that bind to BCR/ABL, a pathogenic fusion protein that causes the chronic phase of Chronic Myelogenous Leukemia (CML), cyclin A, CDK, mitochondria, and the like. Peptides that target the extracellular matrix include peptides that bind fibronectin, fibroblast growth factor, matrix metalloproteinases, prostate specific antigen, cathepsin, and the like.
Cell penetrating peptides include R8, TAT, Transportan and Xentry.
Beta-hairpin peptides, e.g. Z Aβ3 And are useful in the treatment of protein folding diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, cystic fibrosis, gaucher disease and many other degenerative and neurodegenerative disorders.
Immune checkpoint inhibitor beta-hairpin peptides can be identified as high affinity for each other on the surface of immune checkpoint receptorsActive immune checkpoint inhibitor ligand peptides (e.g., surface peptides). For example, surface β -hairpin PD-1 peptides have been identified herein that interact with high affinity with PD-L1. As used herein, high affinity refers to K D Is 0.1-1000 nM. Such peptides may have a length of 5 to 50 amino acids and do not correspond to an entire immune checkpoint inhibitor.
Exemplary β -hairpin PD-1 peptides include:
SEQ ID NO:1：TYLCGAISLAPKLQIKESLRA(βH 1 -wt sequence)
SEQ ID NO:2：TYVCGVISLAPRIQIKESLRA(βH 1 -mutant sequences)
SEQ ID NO:3：VLNWYRMSPSNQTDRKAA(βH 2 -wt sequence)
SEQ ID NO:4：HVVWHRESPSGQTDTKAA(βH 2 -wt sequence)
In one aspect, the β -hairpin peptide comprises a tryptophan zipper. SEQ ID NO:4 is SEQ ID NO: 5.
SEQ ID NO:5：HKVWHWESPSGQWDTWAA(Trp-ZipβH 2 mutant sequences)
As used herein, a tryptophan zipper is a β -hairpin peptide comprising four tryptophan residues located on the same peptide surface that aggregate and stabilize the β -hairpin peptide.
the trpzip strategy (e.g., stabilization of the peptide fold structure), in addition to conjugation to the nanoparticle, further enhances the binding kinetics of the β -hairpin peptide due to the stability of the peptide fold structure on the surface of the nanoparticle (e.g., dendrimer). Intermolecular forces between the peptide and the nanoparticle surface, including hydrogen bonding, van der waals forces, dipolar interactions, also help stabilize the folded structure of the peptide, thereby improving overall binding kinetics.
In addition to the β -hairpin peptide, a large number of end groups on the core of the multivalent nanoparticle can be used for conjugation of a variety of molecules. The multivalent nanoparticle core may be associated, e.g., complexed or conjugated, with one or more therapeutic, prophylactic or diagnostic agents. Diagnostic agents include imaging agents.
In one aspect, the therapeutic agent is a chemotherapeutic agent. TransformingTherapeutic agents include, but are not limited to, the following categories: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other antineoplastic agents. In addition to the chemotherapeutic agents mentioned above, i.e. doxorubicin, paclitaxel, other suitable chemotherapeutic agents include the tyrosine kinase inhibitor imatinib mesylate
Figure BDA0003624602240000161
Or
Figure BDA0003624602240000162
Cisplatin, carboplatin, oxaliplatin, mechlorethamine (mechlorethamine), cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, pyrimidine, vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin (L01CB), etoposide, docetaxel, topoisomerase inhibitors (L01CB and L01XX) irinotecan, topotecan, amsacrine, etoposide phosphate, teniposide, actinomycin D, lonidamine, and monoclonal antibodies (e.g., trastuzumab
Figure BDA0003624602240000163
Cetuximab, bevacizumab and rituximab
Figure BDA0003624602240000164
And so on.
Other examples of therapeutic agents include, but are not limited to, antimicrobial agents, analgesic agents, anti-inflammatory agents, and others. Antibiotics commonly used to treat infections, such as vancomycin, including infections caused by methicillin-resistant staphylococcus aureus (MRSA), may be incorporated into the particles. The particles optionally include cyclosporin, a lipophilic drug acting as an immunosuppressant, widely used after allogeneic organ transplantation to reduce the activity of the patient's immune system and the risk of organ rejection (by the Nowa company under the trade name
Figure BDA0003624602240000165
And
Figure BDA0003624602240000166
sales). Cyclosporin-containing particles may also be used in topical emulsions to treat keratoconjunctivitis sicca. In this regard, particles having multifunctional surface domains incorporating such drugs can be designed to deliver equal doses of each drug directly to cancer cells, thereby potentially minimizing the amount typically delivered to the patient and minimizing collateral damage to other tissues.
Therapeutic agents also include therapeutic nucleic acids, such as gene silencing agents, gene modulators, antisense agents, peptide nucleic acid agents, nuclease agents, RNA agents, and DNA agents. Nucleic acid therapeutics include single-or double-stranded RNA or DNA, particularly RNA, such as triplex oligonucleotides, ribozymes, aptamers, small interfering RNAs including siRNA (short interfering RNA) and shRNA (short hairpin RNA), antisense RNA, microrna (mirna), or portions thereof, or analogs or mimetics thereof, capable of reducing or inhibiting expression of a target gene or sequence. Inhibitory nucleic acids may function, for example, by mediating degradation or inhibiting translation of mRNA complementary to the interfering RNA sequence.
A diagnostic agent is an agent capable of detecting or imaging a tissue or disease. Examples of diagnostic agents include, but are not limited to, radiolabels, fluorophores, and dyes.
Imaging agent refers to a label attached to the random copolymer of the invention for imaging a tumor, organ or tissue of a subject. Examples of imaging agents include, but are not limited to, radionuclides, fluorophores (e.g., fluorescein, rhodamine, isothiocyanates (TRITC, FITC), Texas Red, Cy2, Cy3, Cy5, APC, and
Figure BDA0003624602240000171
(Invitrogen, Carlsbad, Calif.) series fluorophores), antibodies, gadolinium, gold, nanomaterials, horseradish peroxidase, alkaline phosphatase, derivatives thereof, and mixtures thereof.
Radiolabelling refers to nuclides that exhibit radioactivity. "nuclides" refer to atom types specified by their atomic number, atomic mass, and energy state, such as carbon 14 (C: (C) (), (C)) 14 C) In that respect By "radioactive" is meantRadiation emitted by radioactive materials includes alpha particles, beta particles, nuclei, electrons, positrons, neutrinos, and gamma rays.
Administration of a prophylactic agent may occur before symptoms characteristic of the disease or condition are manifested, such that the disease or condition is prevented or alternatively delayed in its progression.
Therapeutic molecules, diagnostic agents, and prophylactic agents can be combined with the multivalent nanoparticle core by chemical conjugation, physical encapsulation, and/or electrostatic interaction methods.
Also included are pharmaceutical compositions comprising the nanoparticle systems described herein. The pharmaceutical composition may further comprise a therapeutic, prophylactic or diagnostic agent as described above.
As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of nanoparticles, as well as pharmaceutically acceptable excipients, such as diluents, preservatives, solubilizers, emulsifiers, and adjuvants. As used herein, "pharmaceutically acceptable excipients" are well known to those skilled in the art.
Tablets and capsules for oral administration may be in unit dosage form and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; a disintegrant, such as potato starch, or an acceptable wetting agent, such as sodium lauryl sulfate. The tablets may be coated according to methods well known in conventional pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters (such as glycerol, propylene glycol or ethanol); preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.
For topical application to the skin, the medicament may be formulated as a cream (cream), lotion (lotion) or ointment (ointment). Cream or ointment formulations which can be used in medicine are conventional formulations well known in the art. Topical administration includes transdermal formulations, such as patches.
For topical application to the eye, the inhibitor may be formulated as a solution or suspension in a suitable sterile aqueous or nonaqueous vehicle. Additives such as buffers, for example sodium metabisulphite or disodium edetate; preservatives, including bactericides and fungicides, such as phenylmercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickeners, such as hypromellose.
The active ingredient may also be administered parenterally (subcutaneously, or intravenously, or intramuscularly, subcutaneously (intramenally), or by infusion techniques) in the form of sterile injectable aqueous or oleaginous suspensions in sterile media. Depending on the vehicle and concentration used, the drug may be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives, and buffering agents can be dissolved in the vehicle.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The term "unit dose" refers to a predetermined amount of active ingredient sufficient to effectively treat a given activity or condition. The preparation of each type of pharmaceutical composition comprises the step of bringing into association the active compound with the carrier and one or more optional auxiliary ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid or solid carriers and then, if necessary, shaping the product into the desired unit dosage form.
In one aspect, a method of making a nanoparticle system includes contacting a multivalent nanoparticle core comprising a plurality of reactive end groups with a composition comprising an immune checkpoint inhibitor under conditions sufficient to conjugate a plurality of immune checkpoint inhibitors to the multivalent nanoparticle core and provide the nanoparticle system. Exemplary terminal groups include coupling linkers and reactive epoxides such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide, 1' -carbonyldiimidazole, N-succinimidyl S-acetylthioacetate, N-succinimidyl-S-acetylthiopropionate, 2-mercaptoethylamine, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succinimidyl iodoacetate, succinimidyl 3- (2-pyridyldithio) propionate, N-hydroxysuccinimide ester, N-hydroxysulfosuccinimidyl ester, N-gamma-maleimidobutyryl-oxysuccinimidyl ester, N-gamma-maleimido-butyloxy sulfosuccinimidyl ester, N-hydroxysuccinimide ester, N-gamma-maleimido-iodoacetate, N-succinimidyl 3- (2-pyridyldithio) propionate, N-hydroxysuccinimide ester, N-gamma-butyloxy-sulfosuccinimidyl ester, and the like, Nitrophenyl esters, tetrafluorophenyl esters, pentafluorophenyl esters, thiopyridyl esters, thionitrophenyl esters, and combinations comprising at least one of the foregoing.
In one embodiment, the multivalent nanoparticle core comprises two or more different types of reactive end groups to enhance the reactivity and/or specificity of the core.
In another embodiment, the immunotherapy method comprises administering to a subject (e.g., a human subject) a nanoparticle system described herein. Exemplary human subjects include cancer patients and patients with immune disorders (e.g., multiple sclerosis and rheumatoid arthritis). The nanoparticles can target the immune system by interacting with T cells, cancer cells, and/or antigen presenting cells.
When the β -hairpin peptide is an immune checkpoint inhibitor peptide, the compositions and methods described herein are applicable to all cancers, including solid tumor cancers, such as breast, prostate, ovarian, lung, and brain cancers, as well as liquid cancers such as leukemias and lymphomas.
The methods described herein may be further combined with additional cancer therapies such as radiation therapy, chemotherapy, surgery, and combinations thereof.
The invention is further illustrated by the following non-limiting examples.
Examples
Example 1: synthesis and analysis of PD-1/PD-L1 peptide inhibitor complex
Materials and methods
And (4) peptide synthesis. Fmoc-amino acids and coupling reagents were purchased from Anaspec (USA) and Novabiochem (Germany), while general chemicals were obtained from Sigma-Aldrich (USA). Rink Amid MBHA resin LL (Novabiochem, germany) was used as a backbone for peptide synthesis. The sequence was synthesized using standard amino acids with standard Fmoc protecting groups. Final deprotection and cleavage of the peptide from the resin involved treating the resin bound peptide with cleavage cocktail (cocktail) (trifluoroacetic acid (TFA): benzylsulfide: Ethanedithiol (EDT) ratio 95:2.5:2.5) for 2 hours followed by precipitation with tert-butyl methyl ether. The peptide was purified using reverse phase HPLC (mobile phase of water/acetonitrile and 0.1% TFA). Peptide molecular weight by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (AXIMA) TM Shimadzu, japan) was quantified using α -cyano-4-hydroxycinnamic acid (CHCA) as a base. Peptide concentration tryptophan (5500M) was used in water/acetonitrile (1:1) by ultraviolet-visible (UV-Vis) spectrophotometry -1 cm -1 ) And tyrosine (1280M) -1 cm -1 ) The molar extinction coefficient at 280nm was quantified.
Preparation of dendrimer-peptide conjugates (DPC). G7 PAMAM dendrimer (10mg) dissolved in 1mL of methanol (Dendritech, USA) was acetylated by the addition of 60, 80 and 90% acetic anhydride corresponding to the number of amine groups on the surface of the dendrimer and more than 600 moles of Triethylamine (TEA) in the dendrimer. The reaction was carried out at room temperature for 24 hours with vigorous stirring. Using Vivaspin TM Turbo 15(MWCO 10,000, Sartorius, Germany) at a speed of 4,000r.p.m. for 15 minutes with ddH 2 O wash 10 times (centrifugal filtration) to remove excess reagent. The acetylated dendrimer was then fluorescently labeled with N-hydroxysuccinimide rhodamine (NHS-RHO) to better quantify and visualize the nanoparticles. The dendrimer was dissolved in dimethyl sulfoxide (DMSO), and 10 equivalents of the amount of dendrimer NHS-RHO dissolved in DMSO were added dropwiseAnd then reacted at room temperature for 24 hours. Excess reagent was removed by centrifugation. The NHS-RHO labeled dendrimer is then conjugated to the peptide. The dendrimer was dissolved in phosphate buffered saline (PBS, pH 7.4), and then 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and NHS dissolved in PBS were added to the dendrimer solution. The solution was then stirred vigorously at room temperature for 1 hour. Thereafter, the peptide dissolved in PBS was added, followed by reaction at room temperature for 24 hours. Excess peptide and reagents were removed by centrifugation.
Surface Plasmon Resonance (SPR). Using BIAcore TM X (Pharmacia Biosensor AB, Uppsala, Sweden) for SPR analysis. Briefly, PD-L1 protein (R)&D systems) were fixed on the surface of the gold film coated with carboxylated dextran of the sensor chip (CM5 sensor chip, GE Healthcare) by EDC/NHS chemistry. 30. mu.L of the sample solution was injected at a flow rate of 20. mu.L/min. The sample solution was allowed to flow into both channels (channel 1 for reference and channel 2 PD-L1), and the final SPR sensorgram was obtained by subtracting the signal from channel 2 from the signal from channel 1.
Circular Dichroism (CD) spectroscopy. The CD spectra were obtained using an Aviv 420 type circular dichroism spectrometer (Aviv, usa). The samples were analyzed from 260 to 190nm using a quartz cuvette with a 1mm path length.
Attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy. Will dissolve in H 2 The sample in O was dried over a ZnSe ATR prism. FTIR spectra were obtained on a Bruker Equinox 55/S FTIR spectrometer.
Molecular Dynamics (MD) simulation studies. The system was simulated using NAMD and CHARMM force fields (CHARMM universal force field and CHARMM36) in an NPT ensemble at 1bar P and 300K using langevin dynamics with a damping constant of 0.01ps-1 and a time step of 2 fs. In the presence of periodic boundary conditions, long range electrostatic interactions were calculated by the PME method. The number of hydrogen bonds is analyzed by VMD, and the truncation distance is
Figure BDA0003624602240000212
The angle is 60 °.
Fluorescence Polarization (FP) assay. In PBSThe FP assay of (d) was used for PD-L1 binding and competition assays (λ ex-480 nm; λ em-535 nm). Fluorescence anisotropy measurements were used in 384-well plates at room temperature
Figure BDA0003624602240000211
M1000 Pro microplate reader (Tecan). After 30 min incubation, the f β H2_ mt/PD-L1 complex was used in the FP competition assay and titrated together with competitors.
And (5) culturing the cells. Human Renal Cell Carcinoma (RCC) cell line 786-O and breast cancer cell line MCF-7 were used as high-and low-expressing PD-L1 cancer cell models, respectively. The human T lymphocyte cell line Jurkat was used in this study as a representative immune cell expressing PD-1. 786-O and Jurkat T cells were cultured in RPMI medium, while MCF-7 cells were grown in DMEM medium. All cell culture media were supplemented with 1% (v/v) penicillin/streptomycin (P/S) and 10% (v/v) Fetal Bovine Serum (FBS). Cells were incubated at 37 ℃ with 5% CO 2 Is incubated in a humid environment.
Western blotting was carried out. Total protein lysates from 786-O and MCF-7 cells were prepared by incubating the cells with RIPA buffer (150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 50mM Tris-HCl (pH 7.5), 2mM EDTA, protease inhibitor cocktail II). By BCA assay (Pierce) TM BCA protein assay kit) the protein content of the lysate was quantified. 25 μ g of protein was separated on a 4-16% gradient acrylamide gel and transferred to PVDF membrane under wet transfer conditions. Primary antibody against PD-L1 (polyclonal anti-human PD-L1, AF156, R) was used at 4 deg.C&D Systems) and primary antibodies against β -actin (monoclonal anti- β -actin, MAB8929, R)&D Systems) were probed overnight and then incubated with the appropriate secondary antibody for 1 hour at room temperature. Using the chemiluminescent reagent Clarity TM Western ECL substrate (Bio-Rad) detection of proteins on the blot and use of Syngene TM Box F3(Syngene, Frederick, MD) imaging.
Assessment of T cell cytokine production. The activity of Jurkat T cells was studied in a cancer-immune cell co-culture system by assessing the amount of interleukin 2(IL-2) secreted by T cells using ELISA. Cancer cells were incubated in 96-well plates (5,000 cells/well) for 48 hours. Cancer cells and T cells were pretreated with interferon-gamma (IFN γ,10ng/mL) and phytohemagglutinin (PHA,1 μ g/mL)/phorbol myristate acetate (PMA,50ng/mL) for 30 hours to activate PD-L1 and PD-1 expression, respectively. Cancer cells were then treated with ICI for 6 hours and subsequently co-cultured with Jurkat T at a ratio of 1: 4. After 48 hours of incubation, cell culture supernatants were collected and evaluated for IL-2.
And (4) measuring chemical sensitivity. Chemosensitivity assays were performed by measuring the synergistic cytotoxic effect of immune checkpoint inhibitors and the chemotherapeutic drug doxorubicin. Cancer cells were seeded in 96-well plates (5,000 cells/well) and incubated for 48 hours. Cancer cells and Jurkat T cells were pretreated with IFN γ and PHA/PMA as described previously. Cancer cells were then stained with calcein AM (1.5 μ M) and then treated with ICIS for 2 hours. Cells were co-cultured at a ratio of 1:4 for an additional 24 hours prior to doxorubicin (5 μ M) treatment. The effect of each ICI on cancer cell survival after doxorubicin treatment (2 hours) was analyzed in terms of changes in fluorescence intensity.
As a result:
to develop the PD-1/PD-L1 peptide inhibitor complex, a β -hairpin peptide from the surface of PD-1 was identified and engineered by a combination of three synergistic methods (fig. 1). First, we have optimized using the amino acid composition of the non-native PD-1 ectodomain to exhibit high PD-L1 affinity. Secondly, these peptides are multivalent conjugated to the dendrimer surface, enabling a synergistic, strong interaction with the various PD-L1 proteins on tumor cells. Third, conjugation on the surface of dendrimers helps peptides fold into their native structure β -hairpins due to the excluded volume effect and dendrimer-peptide interactions.
Given the potential synergistic effects of these engineered approaches, we therefore hypothesized that this DPC strategy would make the peptide superior to native PD-1 in competitive interaction with PD-L1, thereby restoring anti-tumor immunity.
To develop DPCs targeting PD-L1, β -hairpin peptides (β H1_ mt and β H2_ mt, fig. 2) were synthesized based on engineered PD-1 ectodomain sequences and attached to the surface of seventh generation (G7) poly (amidoamine) (PAMAM) dendrimers. Given that the surface area of the G7 PAMAM dendrimer is approximately 10 times greater than the surface area of the PD-1/PD-L1 interface (fig. 3), 90% of the dendrimer amine groups were acetylated to control the amount of attached peptide prior to conjugation. The resulting DPCs were then analyzed using Surface Plasmon Resonance (SPR), designated G7- β H1_ mt and G7- β H2_ mt, to measure their binding kinetics. As shown in FIG. 4, G7-. beta.H 2_ mt had a higher affinity for the immobilized PD-L1 protein than G7-. beta.H 1_ mt, whereas the fully acetylated dendrimer showed no binding reaction. In addition, the PD-L1 affinity of G7- β H2_ mt was also higher than the wild-type β H2-dendrimer conjugate control (G7- β H2_ wt), indicating that the engineered PD-1 sequence (β H2_ mt) resulted in higher affinity. Therefore, we selected β H2_ mt peptides as PDL1 targeting ligands and conjugated them to dendrimer surfaces with different degrees of acetylation to determine effective peptide titers. It is expected that the strength of binding as a result of multivalent binding interactions will be proportional to the number of ligand molecules. However, for DPCs with higher amounts of β H2_ mt peptide (i.e. DPCs prepared from 80% and 60% acetylated dendrimers), lower affinity of PD-L1 was observed (fig. 5). These unexpected results may be attributed to the fact that the optimized spatial distance between ligands plays a key role in achieving stronger binding, rather than merely increasing the number of ligands, which is also observed elsewhere. Together, these results indicate that G7- β H2_ mt, prepared from 90% acetylated dendrimer, is likely to antagonize PD-1/PD-L1 interactions more effectively than its counterparts, G7- β H1_ mt and G7- β H2_ wt.
Next, we compared the PD-L1 binding kinetics of G7- β H2_ mt with anti-PD-L1 (aPD-L1) antibody and free β H2_ mt peptide. SPR analysis showed that G7- β H2_ mt showed five orders of magnitude higher affinity for PD-L1 than β H2_ mt (K) D Is 2.75X 10 -9 And 1.19X 10 -4 ) This was associated with the PD-L1 affinity (K) of the whole aPD-L1 antibody D Is 2.09X 10 -9 ) Rather, as shown in fig. 6-8. Notably, the dissociation rate constant (k) for G7- β H2_ mt compared to the free peptide d ) About 180-fold reduction, although there were only 30 peptides per dendrimer (data not shown)). This non-linear enhancement of binding is characteristic of multivalent binding effects, i.e. multivalent objects have a higher chance of re-binding to the target molecule than their monovalent counterparts (statistical re-binding mechanism). Interestingly, the binding rate constant (k) which plays a minor role in multivalent binding effects is known a ) Also shows a non-linear increase (2.52 multiplied by 10) 5 And 1.07X 10 3 ). This result indicates that, in addition to multivalent binding, other factors also contribute to significantly enhance PD-L1 binding of G7- β H2_ mt.
To elucidate the mechanism behind the enhanced binding kinetics of G7- β H2_ mt, we investigated the folding structure changes of the peptides, which significantly affected their target affinity and selectivity after conjugation to dendrimers [20] . Figure 9 shows a Circular Dichroism (CD) spectrum (red line) for G7- β H2_ mt, where some degree of peptide folding (negative signal at about 220 nm) is observed, unlike the typical broad negative CD band centered at 222nm due to the α -helical structure. In contrast, free β H2_ mt showed an almost unfolded random coil structure (black line), as shown by the strong negative CD band at about 200 nm. Note that the CD spectra (red and grey lines) of the dendrimers omit signals below 218nm because the large number of amide linkages in the dendrimer backbone absorb far Ultraviolet (UV) light. A concentration of 1 μ M of dendrimer was used to minimize absorption of low wavelength light by the macromolecule, but still obtain a sufficiently strong signal for data interpretation in the 190-230nm range that is generally characteristic of the secondary structure of peptides.
We then used attenuated total reflectance-fourier transform infrared (ATR-FTIR) to study the folding behavior of the peptides. As shown in FIG. 10, FTIR spectra confirmed the presence of random coil and β -sheet (1640 cm) in β H2_ mt peptide -1 Nearby broadband), and micro beta-turn structures (1670 and 1690 cm) -1 Nearby weak absorption). In contrast, the FTIR spectrum of G7- β H2_ mt shows the characteristic of the β -hairpin structure, i.e., the inter-strand vibrational coupling at 1634cm -1 Has distinguishable absorption, and beta-turn conformation at 1668 and 1683cm -1 Has distinguishable absorption. Thus, both structural analyses together indicate that the hairpin structure of β H2_ mt is stabilized by dendrimer conjugation. These results are consistent with several theoretical studies, indicating that surface binding stabilizes the biomolecule structure by the excluded volume effect (fig. 11), i.e. in the presence of the substrate, the conformational freedom of the peptide to be unfolded is limited, thereby reducing the entropy cost of folding.
To support experimental results, we also performed Molecular Dynamics (MD) simulations using a single β H2_ mt peptide-five generation (G5) PAMAM dendrimer conjugate. Note that for efficient computation time, G5 PAMAM dendrimers were used instead of the larger G7. The peptide behavior of the original (1) extended and (2) folded β H2_ mt on dendrimer surfaces was compared for 500ns (fig. 12 and 13). In contrast to free β H2_ mt, which exhibits a folded and extended conformation in solution, the initially extended peptide bends into a folded structure and the initially folded β H2_ mt stably maintains the folded conformation on the dendrimer surface. Interestingly, peptides generated various intermolecular forces with the dendrimer surface, including hydrogen bonding, electrostatic interactions, and van der waals interactions, while maintaining hairpin structures (data not shown). In general, it is known that the formation of such molecular interactions with surfaces reduces the structural stability of proteins. However, β H2_ mt was an isolated peptide fragment that was initially exposed to multiple molecular interactions throughout the PD-1 protein structure (data not shown). In addition to the reduced entropy penalty described above, these molecular interactions appear to help further stabilize peptide molecules in a folded conformation on the surface of the dendrimer. The best method of stabilizing the β -hairpin is covalent cross-linking of the two chains in the peptide. However, chemical modifications often complicate the peptide preparation process, leading to a significant reduction in synthetic yield. Introduction of interchain noncovalent bonding is another commonly used strategy; however, it requires a large number of amino acid substitutions, which may affect the physicochemical properties of the peptide. In contrast, our DPC strategy allows stabilization of the hairpin structure of the peptide with minimal modification of the peptide structure. In combination with the multivalent binding advantages conferred by dendritic nanoparticles, this unique DPC platform provides a new approach to effectively antagonize and target β -sheet rich protein surfaces.
Next, we investigated the possibility of using G7-. beta.H 2_ mt as a PD-1/PD-L1 inhibitor. For Fluorescence Polarization (FP) competition assays, fluorescein-conjugated β H2_ mt (f β H2_ mt) peptide was synthesized and used to construct the target complex with PD-L1 protein (fig. 14). In competition experiments (f β H2_ mt, 10 nM; PD-L1, 2 μ M), complex integrity was not affected by the addition of β H2_ mt peptide and fully acetylated dendrimer, whereas G7- β H2_ mt resulted in a dose-dependent displacement of f β H2_ mt from PD-L1 (FIG. 15). Interestingly, despite a slightly lower affinity for PD-L1, DPC showed more potent competition than the aPDL1 antibody, which could be attributed to multivalent ligand display that allowed accommodation of multiple target proteins on the surface of DPC (fig. 16).
To further examine their efficiency, DPCs were then tested in vitro. As shown in fig. 17 and 18, strong cell interaction of G7- β H2_ mt with 786-O cells (PD-L1 overexpressing cell line) was observed using fluorescence microscopy, while interaction of DPC with MCF-7 cells (low expression level of PD-L1) was significantly reduced, indicating that G7- β H2_ mt has high PD-L1 selectivity. In vitro PD-1/PD-L1 inhibition was then assessed by measuring the amount of cytokines (interleukin 2, IL-2) secreted by Jurkat T cells after co-culture with cancer cells, as described elsewhere (fig. 19). It is well known that blocking PD-1/PD-L1 binding activates T cells and promotes cytokine production. Figure 20 shows that G7- β H2_ mt effectively inhibited 786-O/Jurkat T cell interaction, resulting in a 1.52-fold increase in IL-2 secreted by T cells compared to untreated cancer cells (p <0.001), which is even more pronounced than aPD-L1 antibody which only shows a 1.34-fold enhancement (p 0.011). This can be attributed to the multivalent binding effect of G7- β H2_ mt. Note that neither free peptide nor fully acetylated dendrimer induced significant IL-2 production.
To demonstrate the inhibition of PD-1/PD-L1, we also tested whether DPC treatment could affect chemotherapeutic resistance in cancer cells, and in many clinical and preclinical studies immune checkpoint blockade has been shown to reduce this resistance. Co-culture models of tumor (786-O or MCF-7) and Jurkat T cells were used to study the synergistic cytotoxic effects of Doxorubicin (DOX) and G7- β H2_ mt (FIG. 21). Cancer cells treated with different PD-L1 antagonists were co-cultured with T cells and then subjected to DOX treatment to induce cell death. As shown in figure 22, blocking PD-L1 molecules with G7- β H2_ mt significantly reduced chemotherapy resistance of 786-O cells, as compared to cells treated with doxorubicin alone, which was shown to be 8.4 ± 3.8% less viable (p ═ 0.022). G7- β H2_ mt was slightly more potent than aPD-L1 antibody which induced a 7.2 ± 3.7% decrease in cell viability (p ═ 0.030). Considering that the free peptide had little effect on chemotherapy resistance (1.8 ± 2.0% reduction; p ═ 0.334), while the fully acetylated G7 dendrimer had no cytotoxic effect on cancer cells, this result provided a further layer of evidence that multivalent G7- β H2_ mt effectively blocked the PD-1/PDL1 immune checkpoint. MCF-7 cells expressing low levels of PD-L1 also showed a similar trend, although the difference was not as significant as 786-O cells expressing high levels of PD-L1.
In summary, we have demonstrated that the DPC approach enables multimerization and conformational stabilization of β -hairpin peptides isolated from the protein surface on nanoscale dendrimers, exhibiting significantly enhanced target affinity. The enhanced binding kinetics translated into a significant increase in efficiency in vitro, with DPC exhibiting significantly greater PD-1/PD-L1 inhibition than the free peptide and at comparable efficiency levels to aPD-L1 antibody. Inhibition of PD-L1 with antibodies has been clinically proven to be effective in treating a variety of cancer types, such as non-small lung cancer, bladder cancer, and mercker cell skin cancer. However, currently approved monoclonal antibody-based antagonists have limitations due to high cost and lack of modularity. Our strategy has the potential to solve these problems because the dendrimer-peptide system provides a platform technology that can be adapted not only to immunotherapy, but also to other antitumor drugs. In addition, a variety of β -hairpin peptides on the surface of many proteins can be compatible with this DPC approach, increasing their potential in various biomedical applications. This study provides a new engineered peptide-nanoparticle platform for effectively modulating protein interactions to address various diseases, including immune checkpoint blockade for cancer therapy.
Example 2: trpzip DPC
When the β -hairpin was stabilized by Trpzip, the Trp residues were located on the same peptide surface (fig. 25). Thus, surface attachment of the peptides in the proper orientation prevents exposure of the Trp clusters, thereby minimizing their non-specific contribution in target binding. To develop the Trpzip-DPC hybrid system, considering the PD-1/PD-L1 complex structure and the dendrimer conjugation process, the primary structure of pL1 was modified (SEQ ID NO: 4) to provide the amino acid sequence of SEQ ID NO: 5 (fig. 26). Since the amino acid residues buried in the PD-1 core are not involved in PD-L1 binding, we substituted some of them (arginine, aspartic acid and leucine) with Trp residues to introduce Trpzip structure. One of the core residues, valine, was additionally substituted by lysine for dendrimer conjugation, which determined peptide directionality to expose PD-L1 binding surface and to orient the Trp cluster towards the nanoparticle surface (pL1 TZ). As nanoparticle backbone we selected dendrimers.
As shown in FIG. 27, pL1 was found to exist in a random coil structure, with a strong negative band around 200 nm. On the other hand, the CD spectrum of pL1TZ consisted of a negative band at 200nm, a weak shoulder at 215nm (β -sheet), and a weak positive band at 228nm (exciton-coupled band showing interactions between tryptophan residues), which shows the formation of a partially stabilized β -hairpin structure with Trpzip (fig. 28). Interestingly, the secondary structure was even further stabilized upon attachment to the dendrimer surface, as evidenced by the significantly increased CD signal at 200 and 230nm (fig. 29). Based on findings in previous studies, we attributed this surface-assisted hairpin stabilization to the excluded volume effect: the presence of the surface limits the conformational space of the disordered structure, thereby reducing the entropy of the unfolded state (fig. 30). Surface Plasmon Resonance (SPR) analysis showed that the stabilized and multimerized PD-L1 binding peptide exhibited a highly increased PD-L1 binding affinity compared to the free peptide (fig. 31). In addition, DPC also showed significantly enhanced PD-L1 selectivity, hiding tryptophan residues in the peptide/dendrimer interfacial space (fig. 32).
Example 3: dendrimer-peptide conjugates having improved in vitro therapeutic efficacy
The peptide pPD1 has the sequence IYLCGAISLHPKAKIEESPGA (SEQ ID NO:6) that binds to mouse PD-L1. The peptide is conjugated to the dendrimer via its cysteine (C) group by SMCC chemistry.
From Charles RiFemale Balb/c mice (4-6 weeks old) were obtained by ver. Animal procedures and maintenance were performed according to institutional guidelines at the university of wisconsin. The 4T1 cell line was inoculated to the dorsal side of each mouse by subcutaneous injection and tumor volume was measured using calipers. When the tumor volume reaches about 250mm 3 At that time, mice were randomly grouped and treatment was initiated. 100 μ L of the agent was administered by tail vein injection, 3-4 times. Fig. 33 and 34 provide tumor volume and body weight as a function of time. Mice were examined for tumor bioluminescence using the IVIS spectral imaging system (Perkin Elmer). In the data of fig. 35, error bars represent standard errors of the mean.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second, etc. as used herein are not meant to denote any particular order, but rather are used merely for convenience in denoting a plurality, e.g., layers. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are inclusive of the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
SEQUENCE LISTING
<110> Wisconsin school friends research foundation
<120> peptide-nanoparticle conjugates
<130> WIS0054US2 (P200080US02)
<150> US 62/927293
<151> 2019-10-29
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Annotation beta H1-wt sequence
<400> 1
Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Leu Gln Ile Lys
1 5 10 15
Glu Ser Leu Arg Ala
20
<210> 2
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> annotation of beta H1-mutant sequences
<400> 2
Thr Tyr Val Cys Gly Val Ile Ser Leu Ala Pro Arg Ile Gln Ile Lys
1 5 10 15
Glu Ser Leu Arg Ala
20
<210> 3
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Note beta H2-wt sequence, and
<400> 3
Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Arg Lys
1 5 10 15
Ala Ala
<210> 4
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> annotation of beta H2 mutant sequences
<400> 4
His Val Val Trp His Arg Glu Ser Pro Ser Gly Gln Thr Asp Thr Lys
1 5 10 15
Ala Ala
<210> 5
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Trp-Zip beta H2 mutant sequence
<400> 5
His Lys Val Trp His Trp Glu Ser Pro Ser Gly Gln Trp Asp Thr Trp
1 5 10 15
Ala Ala

Claims (31)

1. A nanoparticle system comprising
A multivalent nanoparticle core comprising a plurality of beta-hairpin peptides conjugated thereto.
2. The nanoparticle system of claim 1, wherein the multivalent nanoparticle core comprises a hyperbranched polymer, a dendrimer, a dendron, a mixed nanoparticle, or a micelle.
3. The nanoparticle system of claim 2, wherein the micelle comprises an amphiphilic dendron-coil.
4. The nanoparticle system of claim 2, wherein the mixed nanoparticles comprise a dendrimer-exosome mixture.
5. The nanoparticle system of claim 2, wherein the hybrid nanoparticle comprises a multivalent polymer backbone nanoparticle core having covalently linked thereto a β -hairpin peptide; and a shell encapsulating the polymer-backbone nanoparticle core, wherein the shell comprises a liposome or a polymer shell.
6. The nanoparticle system of claim 2, wherein the dendrimer is a poly (amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a polypropyleneimine (PPI) dendrimer, a diaminobutaneamine polypropyleneimine tetramine (DAB-Am4) dendrimer, a polypropylenylamine (POPAM) dendrimer, a polylysine dendrimer, a polyester dendrimer, a pterene dendrimer, an aliphatic poly (ether) dendrimer, an aromatic polyether dendrimer, or a combination thereof.
7. The nanoparticle system of claim 2, wherein the dendrimer is a PAMAM dendrimer.
8. The nanoparticle system of claim 1, wherein the β -hairpin peptide comprises a tumor targeting peptide, a cell penetrating peptide, a β -hairpin peptide having high affinity for checkpoint inhibitor receptors, or a peptide for treating a protein folding disease.
9. The nanoparticle system of claim 1, wherein the β -hairpin peptide comprises a Trpzip peptide.
10. The nanoparticle system according to claim 1, wherein the β -hairpin peptide is an immune checkpoint inhibitor β -hairpin peptide, such as the surface peptide of PD-1, which binds with high affinity to PD-L1.
11. The nanoparticle system of claim 1, wherein the nanoparticle system is further associated with a therapeutic, prophylactic, or diagnostic agent.
12. The nanoparticle system of claim 11, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic nucleic acid.
13. The nanoparticle system according to claim 11, wherein the diagnostic agent is an imaging agent.
14. A pharmaceutical composition comprising the nanoparticle system of any one of claims 1-13 and a pharmaceutically acceptable excipient.
15. The pharmaceutical composition of claim 17, further comprising a therapeutic, prophylactic, or diagnostic agent.
16. A method of making a nanoparticle system, the method comprising:
contacting a multivalent nanoparticle core comprising a plurality of reactive end groups with a composition comprising one or more β -hairpin peptides under conditions sufficient to conjugate the plurality of β -hairpin peptides to the multivalent nanoparticle core and provide the nanoparticle system.
17. The method of claim 16, wherein the reactive end groups comprise dicyclohexylcarbodiimide, diisopropylcarbodiimide, N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide, 1' -carbonyldiimidazole, N-succinimidyl S-acetylthioacetate, N-succinimidyl-S-acetylthiopropionate, 2-mercaptoethylamine, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succinimidyl iodoacetate, succinimidyl 3- (2-pyridyldithio) propionate, N-hydroxysuccinimide ester, N-hydroxysulfosuccinimidyl ester, N-gamma-maleimidobutyryl-oxysulosuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, N-gamma-maleimido-iodoacetate, N-succinimidyl 3- (2-pyridyldithio) propionate, N-hydroxysuccinimide ester, N-beta-hydroxysuccinimide ester, and mixtures thereof, Nitrophenyl esters, tetrafluorophenyl esters, pentafluorophenyl esters, thiopyridyl esters, thionitrophenyl esters, and combinations comprising at least one of the foregoing.
18. The method of claim 16, wherein the multivalent nanoparticle core comprises two or more different reactive end groups.
19. The method of claim 16, further comprising contacting a multivalent nanoparticle core comprising a plurality of reactive end groups with a therapeutic, prophylactic, or diagnostic agent.
20. The method of claim 16, wherein the multivalent nanoparticle core comprises a hyperbranched polymer, a dendrimer, a dendron, a mixed nanoparticle, or a micelle.
21. The method of claim 20, wherein the micelle comprises an amphiphilic dendron-clew.
22. The method of claim 20, wherein the mixed nanoparticle comprises a dendrimer-exosome mixture.
23. The method of claim 20, wherein the hybrid nanoparticle comprises a multivalent polymer backbone nanoparticle core having covalently linked thereto an immune checkpoint inhibitor; and a shell encapsulating the polymer-backbone nanoparticle core, wherein the shell comprises a liposome or a polymer shell.
24. The method of claim 20, wherein the dendrimer is a poly (amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a polypropyleneimine (PPI) dendrimer, a diaminobutaneamine polypropyleneimine tetramine (DAB-Am4) dendrimer, a polypropylenylamine (POPAM) dendrimer, a polylysine dendrimer, a polyester dendrimer, a pterene dendrimer, an aliphatic poly (ether) dendrimer, an aromatic polyether dendrimer, or a combination comprising one or more of the foregoing.
25. The method of claim 20, wherein the dendrimer is a PAMAM dendrimer.
26. The method of claim 16, wherein the β -hairpin peptide comprises a tumor targeting peptide, a cell penetrating peptide, a β -hairpin peptide having high affinity for a checkpoint inhibitor receptor, or a peptide for treating a protein folding disease.
27. The method of claim 16, wherein the β -hairpin peptide is an immune checkpoint inhibitor β -hairpin peptide, such as the surface peptide of PD-1, which binds with high affinity to PD-L1.
28. The method of claim 16, wherein the β -hairpin peptide comprises a Trpzip peptide.
29. An immunotherapy method comprising administering the nanoparticle system of any one of claims 1-13 to a subject in need thereof.
30. The immunotherapeutic method of claim 29, wherein the subject is a human cancer patient or a human patient with an immune disorder.
31. The immunotherapeutic method of claim 29, further comprising administering radiation therapy, chemotherapy, surgery, or a combination comprising at least one of the foregoing.
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